Cas9 Nickase-Based Genome Editing in Clostridium cellulolyticum

Clostridium cellulolyticum is a model mesophilic, cellulolytic bacterium, with the potential to produce biofuels from lignocellulose. However, the natural cellulose utilization efficiency is quite low and, therefore, metabolically engineered strains with increased efficiency can decrease both the overall cost and time required for biofuel production. Traditional genetic tools are inefficient, expensive, and time-consuming, but recent developments in the use of CRISPR-Cas genetic editing systems have greatly expanded our ability to reprogram cells. Here we describe an established protocol enabling one-step versatile genome editing in C. cellulolyticum. It integrates Cas9 nickase (Cas9n) which introduces a single nick that triggers repair via homologous recombination (SNHR) to edit genomic loci with high efficiency and accuracy. This one-step editing is achieved by transforming an all-in-one vector to coexpress Cas9n and a single guide RNA (gRNA) and carries a user-defined homologous donor template to promote SNHR at a desired target site. Additionally, this system has high specificity and allows for various types of genomic editing, including markerless insertions, deletions, substitutions, and even multiplex editing.